Purification and partial characterization of the phosphoglucomutase isozymes from human placenta

We have developed a simple procedure for the purification of phosphoglucomutase (PGM) isozymes from human placenta of healthy women. The technique involves the ammonium sulfate fractionation, ion-exchange and dye-ligand chromatographies. By this method we obtained homogeneous isozyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci. The final specific activities were 1134.6-1441.8 units/mg for PGM1 forms and 40.2-46.5 units/mg for PGM2 forms. On SDS-polyacrylamide gel electrophoresis analysis, the final preparations gave a single protein band of 58,500 and 69,000 Mr for PGM1 and PGM2 isozymes, respectively. These forms have the same kinetic properties, but from the substrate specificity experiments we have found that PGM2 forms are more effective for catalyzing the phosphoribomutase and glucose 1,6-bisphosphate synthase reaction than PGM1 forms. All these properties are shared by the same isozymes previously isolated from human erythrocytes but in this procedure the use of human placenta for the PGM isozymes purification takes advantage of high specific activity of PGM in the extracts of this tissue as well as obtaining highly homogeneous protein suitable for studies at molecular level.     

Results of haptoglobin types investigations in Hungary

Summary This is report about the PGM and PGD isozymes that are present in sperm cells. 4 isozymes that are controlled by the PGM₁ alleles can be detected regularly. Only one isozyme is identified as a product of the PGM ₂ ¹ gene. The isozymes that are controlled by the PGM₃ locus are detected at regular intervals. PGD patterns differ only slightly from those of red cells.

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Zusammenfassung Es wird über die PGM- und PGD-Isoenzyme, die in Spermatozoen vorkommen, berichtet. Als Produkte der PGM₁-Allele lassen sich regelmäßig 4 Isoenzyme erkennen. Nur ein Isoenzym ist erkennbar, welches dem PGM ₂ ¹ -Gen zugeordnet werden kann. Weiterhin lassen sich regelmäßig die Isoenzyme des PGM₃-Locus erkennen. Die PGD-Muster in Spermapherogrammen ähneln denen aus Erythrocytenhämolysaten.

     

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.     

Differential function of the phosphoglucomutase isozymes PGM1 and PGM2

Summary A total of 13 metabolites thought to be possibly inhibitory were tested for their influence on PGM isozyme activities, each at several different concentrations. The analysis of statistical significance was based on enzyme activities obtained by densitometric measurements of starch gels. Five of the substances were found to inhibit PGM activity, three of which definitely and a further one probably led to a significantly stronger inhibition of the isozymes of the PGM ₂ locus than of PGM ₁ isozymes. They are (1) fructose-1,6-diphosphate, (2) adenosine triphosphate, (3) citrate, and (4) possibly 2,3-diphosphoglycerate. Thus, PGM ₁ isozymes proved to function better in hard or perhaps marginal metabolic conditions. Related evolutionary aspects are discussed.     

Isoelectric focusing of human red cell phosphoglucomutase (PGM1). Phenotype distribution in the population of Tuscany and two hereditary variants

Summary Phosphoglucomutase (PGM₁) phenotypes were determined in a population sample of Tuscany, Italy, by isoelectric focusing. The frequencies observed for the four alleles are: PGM ₁ ¹⁺ =0.6012, PGM ₁ ^1- =0.1059, PGM ₁ ²⁺ =0.2495, PGM ₁ ^2- =0.0434. Two variants were detected and it was possible to study the parentage of both of them. The pedigree of the propositus of the first variant shows that the variant occurs in combination with the common alleles PGM₁ 1+ and PGM₁ 2+ and that it has an autosomal dominant inheritance. The second variant has been shown to be a product of the PGM₂ locus, although its PAGIF pattern is included between 2- and 1+ isoenzymes.     

Population studies on human phosphoglucomutase-1 thermostability polymorphism

Summary The electrophoretic and thermostability polymorphisms of the PGM ₁ locus were examined in about 700 Czechoslovakians (Prague) and 3000 Italians. The Italian sample consisted of individuals from Pavia (Northern Italy), Viareggio and Rome (Central Italy) and Naples (Southern Italy). The eight PGM ₁ alleles, PGM ₁ ^1Str , PGM ₁ ^1Sts , PGM ₁ ^1Ftr , PGM ₁ ^1Fts , PGM ₁ ^2Str , PGM ₁ ^2Sts , PGM ₁ ^2Ftr , PGM ₁ ^2Fts , have been considered as combinations of mutations at three different sites, 1/2 S/F and tr/ts, within the PGM ₁ gene and their frequencies discussed in terms of linkage disequilibrium between these sites. All pairwise differences between the samples were significant except for Pavia-Viareggio and Viareggio-Rome. The frequencies of the PGM ₁ ^ts alleles have been found to range from 0.0981 (Prague) to 0.0546 (Naples) and can be ordered according to a North-South cline.This paper is dedicated to Professor Giuseppe Montalenti in occasion of his 80th birthday     

Evidence for two additional common alleles at the PGM1 locus (Phosphoglucomutase-E.C.:2.7.5.1)

Summary Lysates of erythrocytes, leukocytes, lymphocytes, and extracts of sperms were investigated for the PGM₁ isozymes by three techniques: starch gel electrophoresis, high voltage thin-layer agarose gel electrophoresis, and thinlayer isoelectric focusing on polyacrylamide gel. On starch, only the well known common phenotypes 1, 2-1, and 2 were demonstrable. On agarose, different distances of the two main cathodal bands (a, b) among the phenotypes 2-1 were noted. Furthermore, on agarose, some types considered as homozygous on starch gel had a single, sharp banded pattern, while others were broad and blurred. Optimal separation was achieved by isoelectric focusing on polyacrylamide gel. In 291 leukolysates, 10 different phenotypes were identified. These are considered as gene products of 4 different common alleles at the PGM₁ locus as suggested by preliminary family investigations. In a random population from Hessen these four alleles, had the following frequencies: PGM ₁ ^a1 0.6186, PGM ₁ ^a2 0.1718, PGM ₁ ^a3 0.1426, and PGM ₁ ^a4 0.067. The preliminary designation a1, a2, a3 and a4 was chosen as the assumed polymorphism was demonstrated on acrylamide and agarose. The sum of the frequencies PGM ₁ ^a1 and PGM ₁ ^a3 (the gene products of which have apparently the same electrophoretic mobility on starch) is similar to the frequency of the old PGM ₁ ¹ allele (0.757) in Caucasoids, PGM ₁ ^a2 and PGM ₁ ^a4 have a frequency of 0.2388 corresponding with the frequency of the old allele PGM ₁ ² .     

Investigations on the PGM 1 a polymorphism (phosphoglucomutase-EC 2.7.5.1) by isoelectric focusing

Summary The determination of phosphoglucomutase (PGM₁) phenotypes was performed by isoelectric focusing on samples from 1678 unrelated individuals from Hessen, Germany. Ten common phenotypes are considered as gene products of four alleles at the PGM₁ locus with the following frequencies: PGM ₁ ^a1 =0.6305, PGM ₁ ^a2 =0.1844, PGM ₁ ^a3 =0.1320, and PGM ₁ ^a4 =0.0530. Twenty-two different mating types were observed in 113 families with 202 children. The segregation of the phenotypes in the offspring supports the assumed way of autosomal codominant inheritance. The example of a silent allele (PGM ₁ ⁰ ) as well as a rare variant (PGM ₁ ⁷ ) is reported.     

Isoelectric focusing of human red cell phosphoglucomutase

Summary A total of 637 individuals from the rural village of Keneba in The Gambia, West Africa, have been typed for red cell PGM using isoelectric focusing (pH 5–7) in polyacrylamide gels. Eight different phenotypes have been detected. The frequency of the four alleles at the PGM₁ locus was found to be PGM ₁ ¹⁺ 0.795, PGM ₁ ^1- 0.053, PGM ₁ ²⁺ 0.133, and PGM ₁ ^2- 0.019. A study of the PGM phenotypes in 89 families confirmed the simple Mendelian codominant inheritance of the four alleles. Comparative population data suggest that red cell PGM typing by isoelectric focusing might prove to be a useful genetic marker in anthropological studies.     

Purification and structural properties of isozymes of isocitrate dehydrogenase from the mouse

Summary Cytoplasmic and mitochondrial isozymes of NADP⁺-dependent isocitrate dehydrogenase were purified from kidney and heart tissue of an inbred strain of mice. The cytoplasmic isozyme was purified from kidney of DBA/2J mice by means of a four-step procedure which included affinity chromatography with an 8-(6-aminohexyl)-amino-NADP⁺-Sepharose column. The heart mitochondrial isozyme of DBA/2J mice was purified by a two-step procedure involving the use of 8-(6-aminohexyl)-amino-AMP-Sepharose and 8-(6-aminohexyl)-amino-NADP⁺-Sepharose columns. The specific activity of the homogeneous cytoplasmic and mitochondrial isozymes was 40 units/mg and 45 units/mg, respectively. Native and subunit molecular weights of these two isozymes were determined by chromatography on Sephadex G-100, G-150 and G-200 Superfine and polyacrylamide gel electrophoresis. Both isozymes were found to be dimers with the subunit molecular weight of approximatively 35,000. The sedimentation coefficients were determined to be 5.9 and 6.1 for the mitochondrial and cytoplasmic isozyme, respectively. The amino acid compositions of these two isozymes revealed distinct differences in arginine and proline contents. A modified procedure regarding the use of affinity columns for the purification of the weakly bound enzymes is also discussed.National Institute of Health Visiting Fellow.     

Effects of various metabolites on two phosphoglucomutase allozyme activities from Drosophila melanogaster

The effects of various metabolites on the two most common phosphoglucomutase allozymes (PGM^A and PGM^B) in Drosophila melanogaster have been investigated in vitro. 2,3-Diphosphoglycerate (2,3DPG) inhibited PGM^A and PGM^B to the same degree in the presence of 25 µM glucose-1,6-diphosphate (G1, 6P₂). However a higher concentration of G1,6P₂ partially reversed the inhibition of PGM^A exerted by 2,3DPG, so that in the presence of 150 µM G1,6P₂ the inhibition of PGM^A was half that of PGM^B at pH 6.0. Glycerol-3-phosphate (G3P) had no significant effect at pH 7.4 but exerted an activating effect at pH 6.0 which was more pronounced in the case of PGM^B. ATP, citrate, and fructose-1, 6-diphosphate (F1,6P₂) inhibited both PGM^A and PGM^B. The differences found in vitro between these two allozymes can have a significant impact on in vivo function and, therefore, on the maintenance of PGM polymorphism in experimental populations of D. melanogaster studied in the laboratory.     

Immunological studies on erythrocyte phosphoglucomutase isozymes

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.     

Polymorphism of erythrocyte phosphoglucomutase,adenylate kinase and adenosine deaminase in northern Thailand

Summary Phenotypes of the erythrocyte enzymes phosphoglucomutase (PGM) (n-587), adenylate kinase (AK) (n=695), and adenosine deaminase (ADA) (n=616) were determined by horizontal starch gel electrophoresis in Thai subjects from norther Thailand, mainly from the provinces of Chiang Mai and Lamphun. The following gene frequencies were calculated: PGM ₁ ¹ 0.7385 PGM ₁ ² 0.2487 PGM ₁ ⁶ 0.0102 PGM ₁ ⁷ 0.0026, AK ¹ 0.9950 AK ² 0.0050, ADA ¹ 0.9180 ADA ² 0.0820.The regular, apparently autosomal transmission of the PGM ₁ ⁶ and PGM ₁ ⁷ alleles was demonstrated in 7 families revealing sufficient data.

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Zusammenfassung Die Phänotypen der Erythrocytenenzyme Phosphoglucomutase (PGM) (n=587), Adenylatkinase (AK) (n=695), and Adenosindeaminase (ADA) (n=616) wurden mittles horizontaler Stärkegelelektrophorese bei Thailändern aus Nordthailand, hauptsächlich aus den Provinzen Chiang Mai und Lamphun, bestimmt. Auf Grund der Ergebnisse wurden die in der englischen Zusammenfassung angegebenen Genfrequenzen berechnet. Die regelmäßige, anschinend autosomale Vererbung der Allele PGM ₁ ⁶ und PGM ₁ ⁷ wurde in 7 Familien mit ausreichenden Daten nachgewiesen.

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Established and supported by Stiftung Volkswagenwerk.     

A new hereditary variant of the PGM1 erythrocyte enzyme system determined by isoelectric focusing

Summary A new variant of the PGM _a ¹ erythrocyte enzyme system not identical with the known variants of the system has been detected in the hemolyzed red blood cells of a healthy blood donor by isoelectric focusing. Using this technique the variant is represented by two bands, a strong and slow one more cathodically located than the a3 band and a weak one in the position of the a2 band. Using agarose thinlayer or acetate foil electrophoresis the variant is represented only by a minimal cathodic broadening of the PGM₁ 1 band and therefore it is easily overlooked. Investigation of the propositus' family shows that the variant occurs combined with the common alleles PGM ₁ ^a1 , PGM ₁ ^a2 , and PGM ₁ ^a3 and that it has an autosomal dominant inheritance. Obviously the variant has a very low frequency.     

Substrate affinity in PGM1, PGM2, and PGM3 isozymes

Summary An easy method for routine detection of PGM₁, PGM₂, and PGM₃ isozymes is given. Differences in substrate affinity are discussed. Gene products pgm₁ can be differentiated from gene products pgm₃ by cofactor requirement.     

Hominoid triosephosphate isomerase: Characterization of the major cell proliferation specific isozyme

Summary Proliferating cells derived from hominoid species contain electrophoretically separable forms of triosephosphate isomerase (TPI), including a constitutive isozyme and major and minor cell proliferation specific isozymes. Genetic studies have shown that the constitutive and inducible isozymes are products of the same structural gene. A procedure has been developed for the rapid isolation of the constitutive and major proliferation specific TPI isozymes from human lymphoblastoid B cells. [35^S]methionine labeled isozymes were purified through several steps of polyacrylamide gel electrophoresis in sufficient quantities for turnover studies and preliminary structural analysis. The intact isozymes were subjected to 23 steps of automated Edman degradation; both preparations yield a [³⁵S] PTH-methionine only at cycle 14, as expected if the protein is TPI. Neither isozyme contains an blocked NH₂-terminus and length heterogenity at the amino terminal does not exist. A comparison of the two purified isozymes on 2-D PAGE confirms that the constitutive isozyme consists of only type 1 subunits while the major proliferation specific isozyme is composed of a type 1 subunit and a unique type 2 subunit. The type 1 and type 2 subunits differ by at least four charge units under native, nondenaturing conditions of electrophoresis but do not differ in molecular mass. The difference between the type 1 and type 2 subunits is covalent, as the difference in isoelectric point between the two subunits is stable to both 2% SDS and 8 M urea. The expression of TPI-2 does not correlate with the existence of the labile asparagine residues. Turnover studies indicate that the level of each subunit is regulated by differences in rates of synthesis rather than degradation but a precursor-product relationship between the subunits was not observed. Thus the mechanism for synthesis of TPI-2 must operate either during mRNA processing or nascent peptide synthesis and then only in cells from hominoid species.     

Phosphoglucomutase (EC 2.7.5.1.) and adenylate kinase (EC 2.7.4.3.) typings in Koreans and Irish

Summary PGM₁ and AK phenotypes were determined in samples from Korea and Ireland. the frequencies of PGM ₁ ¹ genes amount to 0.916 in Koreans and 0.864 in Irish. AK¹ frequencies come to 0.933 in Koreans and 0.873 in Irish.Supported by the Deutsche Forschungsgemeinschaft.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

The phosphoglucomutase (PGM1)-groups in the Swiss population

Summary The distribution of the phosphoglucomutase(PGM₁)-groups was studied on blood samples obtained from 2638 Swiss adults. The distribution was found to be in excellent agreement with the Hardy-Weinberg equilibrium. The obtained gene frequencies were similar to those observed in other Caucasian populations (PGM ₁ ¹ =0.7586, PGM ₁ ² =0.2414). In 942 mother/child pairs no theoretical impossible combinations were found. No significant difference was observed between the gene frequencies of men and of women. An unusual phenotype, probably 3-1, was found in blood samples from 3 unrelated adults (1 woman and 2 men). In addition 2 children (a child of the woman and a child of one of the men) were found to have this rare phenotype.

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Zusammenfassung An einem Untersuchungsgut von 2638 Blutproben von schweizerischen Erwachsenen wurde die Verteilung der Phosphoglucomutase(PGM₁)-Gruppen untersucht. Die gefundene Verteilung ist in ausgezeichneter Übereinstimmung mit dem Hardy-Weinberg-Gesetz. Die Frequenzen stimmen mit denen anderer kaukasischer Bevölkerungen überein (PGM ₁ ¹ =0,7586, PGM ₂ ¹ -0,2414). In 942 Mutter/Kind-Paaren wurden keine theoretisch unmögliche Kombination gefunden. Es bestand keine signifikante Differenz zwischen den Genfrequenzen von Frauen und Männern. Bei 3 nichtverwandten Personen (1 Frau, 2 Männer) wurde ein seltener Phänotyp (wahrscheinlich 3-1) beobachtet. Der gleiche Typ wurde bei 2 Kindern gefunden (eines war das Kind der Frau, das andere dasjenige von einem der Männer).

     

Genetic markers in Malaysians: Variants of soluble and mitochondrial glutamic oxaloacetic transaminase and salivary and pancreatic amylase,phosphoglucomutase III and saliva esterase polymorphisms

Summary Malaysians of Malay, Chinese, and Indian ancestries were electrophoretically phenotyped for Amy₁ and saliva esterase region 1(Set-1) from saliva, Amy₂ from plasma, soluble and mitochondrial GOT and PGM ₃ from leukocyte and placenta. Kadazans and Bajaus, the indigenous people of Sabah, East Malaysia were surveyed for Amy₂. Three types of variants were observed for Amy₁, one type for Amy₂. Only Indians were found to be polymorphic for Amy₁. Two GOT _s 2-1 and three GOT _m 2-1 variants were found among 281 Chinese while three GOT _m 2-1 variants were found among 311 Malays.Malaysian Malays, Chinese, and Indians were found to be polymorphic for Set-1 and PGM ₃. The gene frequencies in Malays are Set-1F=0.601±0.021, Set-1S=0.399±0.021; PGM ₃ ¹ =0.788±0.020, PGM ₃ ² =0.212±0.020; in Chinese Set-1F=0.497±0.028, Set-1S=0.503±0.028; PGM ₃ ¹ =0.745±0.024, PGM ₃ ² =0.255±0.024; in Indians, Set-1F=0.449±0.031, Set-1S=0.551±0.031; PGM ₃ ¹ =0.755±0.029, PGM ₃ ² =0.245±0.029.